The nuclear function of the heterodimeric NF-kB transcription factor is regulated in part through reversible acetylation of its RelA subunit. We now demonstrate that the p300 and CBP acetyltransferases play a major role in the in vivo acetylation of RelA, principally targeting lysines 218, 221 and 310 for modi®cation. Analysis of the functional properties of hypoacetylated RelA mutants containing lysine-toarginine substitutions at these sites and of wild-type RelA co-expressed in the presence of a dominantly interfering mutant of p300 reveals that acetylation at lysine 221 in RelA enhances DNA binding and impairs assembly with IkBa. Conversely, acetylation of lysine 310 is required for full transcriptional activity of RelA in the absence of effects on DNA binding and IkBa assembly. Together, these ®nd-ings highlight how site-speci®c acetylation of RelA differentially regulates distinct biological activities of the NF-kB transcription factor complex.

The NF-B/Rel family of transcription factors plays a key role in regulating inflammatory and immune responses and other programs of cell growth and survival. The five known mammalian Rel genes encode seven Rel-related proteins: RelA/p65; p105 and its processing product, p50; p100 and its processing product, p52; c-Rel; and RelB. Each contains an N-terminal Rel homology domain (ϳ300 amino acids) that mediates DNA binding, dimerization, and interaction with the IB family of NF-B/Rel inhibitors. RelA, c-RelA, and RelB contain C-terminal transactivation domains, but p50 and p52 do not. Each NF-B/Rel protein forms different homo-or heterodimers with other members of the family, which may contribute to the activation of specific target genes (1, 5).The prototypical NF-B complex is a p50/RelA heterodimer. NF-B is largely sequestered in the cytoplasm through its association with an IB inhibitor. Nuclear NF-B expression is induced by various stimuli, including proinflammatory cytokines, growth factors, DNA-damaging agents, and viral proteins (13). The activation of NF-B can be divided into two phases. The first phase involves cytoplasmic events culminating in the activation of the IB kinases (IKK1 and IKK2). These kinases promote N-terminal phosphorylation of serines 32 and 36 in IB␣, leading to its polyubiquitylation and proteasome-mediated degradation. The liberated NF-B complex rapidly translocates to the nucleus, ending the first phase (13). The second phase occurs primarily in the nucleus and involves posttranslational modification of the NF-B transcription factor complex or relevant histones surrounding NF-B target genes (5). These modifications determine both the strength and duration of the NF-B-mediated transcriptional response (5).One of the nuclear events is the reversible acetylation of RelA (4). Endogenous RelA is acetylated in a stimulus-coupled manner after activation of cells with tumor necrosis factor alpha (TNF-␣), phorbol myristate acetate, or other stimuli at multiple sites, including lysines 122, 123, 218, 221, and 310 (4, 17). The acetyltransferases p300 and CBP appear to play a major role in the in vivo acetylation of RelA (6,17). Sitespecific acetylation of RelA regulates discrete biological actions of the NF-B complex (5, 6). For example, acetylation of lysine 221 by p300/CBP increases the DNA binding affinity of RelA for the B enhancer and, together with acetylation of lysine 218, impairs assembly of RelA with newly synthesized IB␣, which shuttles in and out of the nucleus. Acetylation of lysine 310 does not modulate DNA binding or IB␣ assembly but markedly enhances the transcriptional activity of NF-B. Deacetylation of lysine 310 by histone deacetylase 3 (HDAC3) or SIRT1 inhibits the transcriptional activity of RelA and augments cellular apoptosis in response to 32). While it is clear that signal-coupled acetylation of RelA participates in the nuclear regulation of NF-B action (4, 17), many unanswered questions remain. Chief among these is how the acetylation of RelA is regulated.

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Apoptosis induced by p53 has been proposed to involve activation of the transcription factor NF-B. Here we describe the novel molecular mechanism through which p53 and DNA-damaging agents activate NF-〉. NF-B induction by p53 does not occur through classical activation of the IB kinases and degradation of IB␣. Rather, p53 expression stimulates the serine/threonine kinase ribosomal S6 kinase 1 (RSK1), which in turn phosphorylates the p65 subunit of NF-B. The lower affinity of RSK1-phosphorylated p65 for its negative regulator, IB␣, decreases IB␣-mediated nuclear export of shuttling forms of NF-B, thereby promoting the binding and action of NF-B on cognate B enhancers. These findings highlight a rather unusual pathway of NF-B activation, which is utilized by the p53 tumor suppressor.

The proto-oncogene Cot/Tpl-2 encodes a MAP3K-related serine-threonine kinase. Expression of wild type Cot activates the IkappaB kinases (IKK) leading to induction of NF-kappaB. Conversely, expression of kinase-deficient Cot inhibits CD3/CD28 but not TNF alpha induction of NF-kappaB. These findings suggest the selective involvement of Cot/Tpl-2 or a closely related kinase in the CD3/CD28 costimulatory pathway leading to induced nuclear expression of NF-kappaB. In contrast, a kinase-deficient mutant of the NF-kappaB-inducing kinase (NIK) inhibits both CD3/CD28 and TNF alpha signaling, indicating that these pathways converge at or prior to the action of NIK. Consistent with such a sequential function of these two kinases, Cot physically assembles with and phosphorylates NIK in vivo.

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NF-B corresponds to an inducible eukaryotic transcription factor complex that is negatively regulated in resting cells by its physical assembly with a family of cytoplasmic ankyrin-rich inhibitors termed IB.Stimulation of cells with various proinflammatory cytokines, including tumor necrosis factor alpha (TNF-␣), induces nuclear NF-B expression. TNF-␣ signaling involves the recruitment of at least three proteins (TRADD, RIP, and TRAF2) to the type 1 TNF-␣ receptor tail, leading to the sequential activation of the downstream NF-B-inducing kinase (NIK) and IB-specific kinases (IKK␣ and IKK␤). When activated, IKK␣ and IKK␤ directly phosphorylate the two N-terminal regulatory serines within IB␣, triggering ubiquitination and rapid degradation of this inhibitor in the 26S proteasome. This process liberates the NF-B complex, allowing it to translocate to the nucleus. In studies of NIK, we found that Thr-559 located within the activation loop of its kinase domain regulates NIK action. Alanine substitution of Thr-559 but not other serine or threonine residues within the activation loop abolishes its activity and its ability to phosphorylate and activate IKK␣. Such a NIK-T559A mutant also dominantly interferes with TNF-␣ induction of NF-B. We also found that ectopically expressed NIK both spontaneously forms oligomers and displays a high level of constitutive activity. Analysis of a series of NIK deletion mutants indicates that multiple subregions of the kinase participate in the formation of these NIK-NIK oligomers. NIK also physically assembles with downstream IKK␣; however, this interaction is mediated through a discrete C-terminal domain within NIK located between amino acids 735 and 947. When expressed alone, this C-terminal NIK fragment functions as a potent inhibitor of TNF-␣-mediated induction of NF-B and alone is sufficient to disrupt the physical association of NIK and IKK␣. Together, these findings provide new insights into the molecular basis for TNF-␣ signaling, suggesting an important role for heterotypic and possibly homotypic interactions of NIK in this response.The eukaryotic NF-B/Rel family of transcription factors plays an essential role in the regulation of both inflammatory and immune responses (2, 3). One of the distinguishing characteristics of the NF-B/Rel transcription factor is its posttranslational regulation through interactions with a series of cytoplasmic inhibitory proteins termed IB. IB␣ corresponds to the major IB species bound to the prototypic NF-B (p50-p65) heterodimeric complex. A variety of signals induce nuclear expression of NF-B, including the proinflammatory cytokines tumor necrosis factor alpha (TNF-␣) and interleukin-1␤ (IL-1␤), bacterial lipopolysaccharide, phorbol myristate acetate, CD3 and CD28 costimulation, phosphatase inhibitors such as okadaic acid and calyculin, and various viral proteins, including human T-cell leukemia virus type 1 (HTLV-1) Tax (for a review, see references 34 and 37). These stimuli lead to the phosphorylation of IB␣ on serines 32 and 36 (4, 5, 33, 36...

The major immunoregulatory effects of vasoactive intestinal peptide (VIP) are mediated by structurally distinct type I (VIPR1) and II (VIPR2) G protein-associated receptors on many different types of immune cells. VIP is released in functionally relevant concentrations during many immunologic and inflammatory responses. Mast cells (VIPR1), macrophages (VIPR1 and VIPR2), B cells, and T cells (VIPR1, VIPR2, or VIPR1 and VIPR2) recognize and respond to VIP in patterns that are controlled by the relative levels of expression of VIPR1 and VIPR2. VIPR2 transduces human T-cell chemotaxis, expression of matrix metalloproteinases (MMPs) 2 and 9 and consequently basement membrane and connective tissue transmigration, while signaling suppression of proliferation and cytokine production. In contrast, VIPR1 fails to transduce T-cell chemotaxis but mediates suppression of chemotaxis and MMP expression elicited by some cytokines and chemokines. The relative representation of each type of VIPR, which is presumed to be under cytokine control, thus may determine T-cell responses to VIP and other immune mediators in tissue compartments innervated by VIPergic nerves.